Members of the Ly-49 gene family encode type-II integral transmembrane proteins and are primarily expressed on the surface of murine NK cells. Our monoclonal antibody (4D11) reacts with Ly-49G2 represents a type II transmembrane protein of 268 amino acids with its carboxyl end exposed extracellularly. Ly-49G2+ cells failed to lyse several H-2Dd expressing tumor targets and Con A lymphoblasts from BALB/c, but not C57BL/6 mice. This inhibition of lysis by Ly-49G2+ NK cells was negated by addition of monoclonal antibody (mAb) 4D11 or anti-class I antibodies to Dd or Ld. Our recent studies have generated and characterized a mAb (12A8) that can recognize the Ly-49D subset of murine NK cells. Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 failed to demonstrate an inhibitory pattern when tested on targets [tumor and lymphoblast] expressing a variety of H-2 haplotypes. More importantly we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of Fc-gammaR+ target cells in a RADCC-type assay and induces apoptosis in Ly-49D+ NK cells. Examination of the cytoplasmic domain of Ly-49D indicates that it does not contain the V/IxYxxL ITIM motif found in Ly-49A, C or G2 that has been characterized in the human p58 KIRs. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function. These studies are critical to the study of the structure/function of the various Ly-49 receptors. We have cloned and analyzed a 7 kB mRNA in NK cells that has been termed the NK-TR. Antibodies to the NK-TR or its peptides have indicated a role for this molecule in the cytolytic process of NK cells. Direct evidence for the importance of NK-TR in NK killing was obtained by generating antisense NK-TR transfectants of the rat NK-cell line, RNK-16. Introduction of a cDNA vector producing antisense NK-TR mRNA abolished the ability of these cells to kill several tumor targets as well as virus-infected [NK-sensitive] cell lines. Lectin-mediated killing ADCC and reverse-ADCC were unaffected by antisense expression. We are currently determining if there are alterations in kinase activity in the antisense transfectants in order to identify potential lesions in the normal signal transduction pathway.